Extended Data Figure 4. Cullin 3SPOP promotes PD-L1 ubiquitination and subsequent degradation largely through interaction with the cytoplasmic tail of PD-L1.
a, A schematic illustration of PD-L1 with N-terminal signal peptide, extracellular domain, trans-membrane domain, cytoplasmic tail and the potential SPOP-binding motif in PD-L1.
b, d, Immunoblot (IB) analysis of whole cell lysates (WCL) and GST pull-down precipitates derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting.
c, IB analysis of WCL derived from PC3 stably expressing shCullin 3.
e, g, IB analysis of WCL and immunoprecipitation (IP) derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting.
f, IB of WCL and Ni-NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 hours before harvesting and lysed in the denature buffer.
h, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting.
i, IB of WCL derived from MDA-MB-231 PD-L1 KO cells stably expressing PD-L1 WT, delta 283-290, T290M as well as EV as a negative control.
j, IB analysis of WCL derived from 293T cells transfected with HA-PD-L1 WT and the T290M mutant, which were treated with cycloheximide (CHX) for indicated time points before harvesting.
k, IB of WCL and Ni-NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 hours before harvesting and lysed in the denaturing buffer.
l, IB of WCL derived from 293T cells transfected with indicated constructs.